There is a contamination control standard for a bio-cleanroom indoor environment that has been provided under the pharmacopeia for pharmaceutical manufacturing facilities and it is required to maintain to be less than one bacterium (CFU: Colony-Forming Unit) in a safety cabinet and to be less than 10 CFU in an neighborhood area thereof per 1 m3 of air. Here, CFU is a unit that indicates the number of living bacteria (viable bacteria). In addition, there is also a contamination control standard that has been provided under the pharmacopeia for sterile water (water for use in pharmaceuticals) in the pharmaceutical manufacturing facilities, it is required to operate to be less than 10 CFU/100 mL in level water for injection, and a culture method is used for examinations.
However, in the culture method, since a nutrient agar is cultured for 2-3 days or 10 or more days depending on the kind of a bacterial cell used in an incubator and the number of generated colonies is visually counted, the time is taken until a result is obtained. Under such a background, it is desired to develop a rapid measurement method for a contamination monitor and there are a method of detecting metabolism activity when the viable bacteria grow, a method of detecting it as light by utilizing substances in the bacterial cell and so forth.
Since an Adenosine triphosphate (ATP) bioluminescence method (ATP method) of optically detecting it by utilizing the substances in the bacterial cell requires no culturing process, the result is obtained in one hour even when the time taken for sample preparation is included. If it becomes possible to grasp a microbial contamination condition in one hour, check of a line and products (including intermediates) and measures taken for them will be promoted also during work shift for manufacture and it is expected that a safety management system and a shipment system will be remarkably improved.
In the ATP method, the number of ATPs in a cell is measured by converting it into an amount of light by utilizing a luminescent reaction of fireflies. A principle thereof is such that a luciferase enzyme is made to capture substrate luciferin and ATP molecules and an amount of luminescence when luciferin (oxyluciferin that has been oxidized with consumption of the ATPs shifts from an excited state to a ground state is measured.
At this time, since consumption of one ATP molecule is equivalent to generation of one photon, the number of generated photons is proportional to the number of ATPs. Since the ATP molecules equivalent to 1 attomole (amol=10−18 mol) are present in the viable bacteria as an energy source, the total number of the viable bacteria contained in a measurement sample can be estimated. Further, because of its quantum efficiency (ΦSL: ≠0.5) that is the most excellent in bioluminescence and chemiluminescence, one cell can be detected as the photons equivalent to hundreds of thousands of ones and thus it is the method capable of detecting light equivalent to one cell by the luminescent reaction in principle.
However, it is reported that a detection lower limit of the ATP method is generally about 102 amol (amol=10−18 mol) due to fluctuations in data depending on the performance of a measuring device used and influenced by mixing of the ATP and the bacterial cell present in the environment. As a method of preventing these fluctuations in data, there have been recently reported a dispensing system that possesses a cleaning function for preventing external contamination and a bioluminescence detection system that a highly sensitive light detector has been installed in a space that is light shielded and suppressed in contaminant from the outside in the same device as disclosed, for example, in Patent Literature 1 and it becomes possible to measure the amount of the ATP molecules equivalent to 1 amol.
In addition, in order to improve the performance of the measuring device, there is adopted a means for reducing random noise components and the number of dark pulse counts so as to suppress fluctuations in signal component and extracting the signal component of weak light with high reliability so as to improve detection sensitivity and, for example, in Patent Literature 2, there is disclosed a method of performing temperature control by covering the light detector with a cooling device.